The present invention relates to the development of systems that permit the analysis, validation and rapid throughput of intracellular and extracellular targeting of cells and tissues cultured in a microenvironment.
The ability to study complex or voluminous biological systems with a rigorous experimental model would be useful in a variety of medical industries. The pharmaceutical industry, for example, relies on High Throughput Screening (HTS) of libraries of chemical compounds to find drug candidates. HTS is a method where many discrete compounds are tested in parallel so that large numbers of test compounds are screened for biological activity simultaneously or nearly simultaneously.
The need in biotechnology for automation and miniaturization of components and reagent consumption is elevating interest in micro-fluidics, particularly the need for physically small pumps, valves, and mixing chambers. Microfabricated xe2x80x9clab-on-a-chipxe2x80x9d instruments are emerging for conducting electrophoresis, radiography, protein sequencing, DNA diagnostics, and genotyping that require sample and reagent delivery systems capable of regulating volumes in the 10-1000 nanoliter range.
Miniature biosensors and drug delivery systems are other arenas requiring microfluidic pumps, valves, pipes, and vessels. Micromachined peristaltic pumps may be provided and arranged to deliver a reagent from a reservoir and a sample liquid specimen from a supply source through micro-machined delivery channel sections to a reaction chamber. The reaction chamber may output to a detector.
The present invention provides methods and apparatus for cell culturing in a microenvironment. The environment allows cell culturing in an array format. The invention allows for either introducing cells via microfluidics or printing of cells directly on to the bottom plate which is attached to the controlled microenvironment. A variety of cells could be cultured in the various arrays. The various cells are cultivated with nutrient flow in and out of each culture well. The present invention combines Microfluidics with wafer bonding and attachment to obtain the 3-D microfluidic channel network.
The present invention is useful for analyzing and quantifying several molecular targets within a sample substance using an array having one or more cells, or small molecules applied on a thinfilm substrate which is attachable to the controlled microenvironment. The invention may be used with microarrays in microenvironment for diagnostics, drug discovery and screening analysis, gene expression analysis, cell sorting, microorganic monitoring, micropatterned cell culturing and microsynthesis. Various types of arrays are possible. The following are some of the examples.
One type of array is micropatterned co-cultures. Photolithography is used to pattern biomolecules on glass substrates to create micropattern of cultures of two or more distinct cell types, for example, patterns of hepatocytes and fibroblasts. In this arrangement, cell to cell interactions can be studied based on the specific cell to cell geometry pattern. This geometry pattern can be varied systematically on a glass substrate and this substrate can be attached to the rest of the chip structure for perfusion and analysis. Patterning of cells can also be done by microcontact printing, photobiochemistry and by using digital micromirrors.
The invention can be used in high throughput screening (HTS) strategies designed to identify potential pharmaceuticals from a library of chemical compounds. The devise will enable the investigator to control the precise application (concentration, time of delivery, and delivered volume) of multiple compounds into a microarray of chambers, each containing a parallel culture of viable cells, and simultaneously monitor the real-time effects of that application on various biological processes defined by the user.
The ability to deliver fluids into the culture environment through multiple channels further enables the user to employ both homogenous and heterogeneous assay protocols using the various fluorescent, calorimetric, and bioluminescent reporter systems currently available. Fluorescently labeled biomolecules such as proteins, phospholipids, nucleic acid probes, fluorescent tagged antibodies and synthetic fluorescent reagents with specific binding properties have long been used as reporter molecules to determine the location, amount and chemical environment of subcellular targets and biomolecules.
Such assays are routinely used to monitor the transcriptional activity of a genetic locus throughout the course of an experiment, protein-protein interactions, receptor-ligand interactions, enzymatic activity, basic cell processes such as DNA synthesis and cell division, transmembrane fluxes, pH fluctuations, synthesis of metabolites and countless other biological processes. Furthermore, the ability to transport and segregate viable cells into discrete micro-chambers will further enable the investigator to construct an array of different cell types and simultaneously evaluate the effects of a particular compound or set of compounds on a predefined biological process. For example, the ability of a putative drug to block cell proliferation may be tested on several types of tumor cell lines as well as normal cells at the same time.
Moreover, the device disclosed herein may be used to determine the pharmacokinetic properties of a particular drug on multiple cell types, each cultured in parallel. The invention is particularly suited for tissue engineering applications, which involve the growth and controlled differentiation of, e.g., stem cell lines. Such cells can be cultured in the micro-chambers and treated with a variety of compounds designed to induce differentiation into a particular cell type. Once the appropriate treatment regimen has been established, the device may even be used as a bioreactor to manufacture differentiated cells for therapeutic use.